Interest in the biological control of insect pests has arisen as a result of disadvantages of conventional chemical pesticides. Chemical pesticides generally affect beneficial as well as nonbeneficial species, and insect pests tend to acquire resistance to such chemicals. Furthermore, chemical residues pose environmental hazards and possible health concerns. Biological control presents an alternative means of pest control which can reduce dependence on chemical pesticides.
Baculoviruses are a large group of evolutionarily related viruses which infect only arthropods [Miller, L. K. (1981) in Genetic Engineering in the Plant Sciences, N. Panopoulous, (ed.), Praeger Publ., New York, pp. 203-224; Carstens, (1980) Trends in Biochemical Science 52:107-110; Harrap and Payne (1979) in Advances in Virus Research, Vol. 25, Lawfer et al. (eds.), Academic Press, New York, pp. 273-355, Granados, R. R. and Federici, B. A. eds. (1986) The Biology of Baculoviruses, Vol. 1, Biological Properties and Molecular Biology, CRC Press Inc., Boca Raton, Fla.). Some baculoviruses only infect insects which are pests of commercially important agricultural and forestry crops. Other baculoviruses are known which specifically infect other insect pests, e.g., mosquitoes and fleas. Such baculoviruses are potentially valuable as biological control agents. A potential advantage of baculoviruses as biological pesticides is their host specificity. Because individual baculovirus strains usually only infect one or a few species of insects, they pose little or no risk to man or the environment, and can be used without adversely affecting beneficial insect species.
Baculovirus subgroups include nuclear polyhedrosis viruses, now called nucleopolyhedroviruses (NPVs) and granulosis viruses, now called granuloviruses (GV). In the occluded forms of baculoviruses, the virions (enveloped nucleocapsids) are embedded in a crystalline protein matrix. This structure, referred to as an occlusion body, is the form found extraorganismally in nature, and it is generally responsible for spreading the infection between insects. The characteristic feature of the NPVs is that many virions are embedded in each occlusion body, which is relatively large (up to 5 micrometers). Occlusion bodies of SNPVs (single nucleopolyhedrosis viruses) are smaller and contain a single virion with multiple nucleocapsids each. Multiple nucleopolyedrosis viruses (MNPVS) have multiple nucleocapsids per virion and multiple virions per occlusion body. Granulosis viruses (GVs) have a single virion with one nucleocapsid per occlusion body. The crystalline protein matrix of the occlusion bodies of these forms is primarily composed of a single 25 to 33 kDa polypeptide which is known as polyhedrin or granulin. Groner et al. in The Biology of Baculoviruses, Vol. 1, supra, which is incorporated by reference herein, in Chap. 9, Tables 2 and 7 provides an extensive list of NPV hosts and GV hosts.
In nature, infection is initiated when an insect ingests food contaminated with baculovirus particles, typically in the form of occlusion bodies. The occlusion bodies dissociate under the alkaline conditions of the insect midgut, releasing the virions which then invade epithelial cells lining the gut. Pre-occlusion bodies are also infective (WO 97/08297, published Mar. 6, 1997). Within a host cell, the baculovirus migrates to the nucleus where replication takes place. Initially, specific viral proteins are produced within the infected cell via the transcription and translation of so-called "early genes." Among other functions, these proteins are required for the replication of the viral DNA, which begins 4 to 6 hours after the virus enters the cell. Viral DNA replication proceeds up to about 24 hours post-infection (pi). From about 8 to 24 hours pi, infected cells express "late genes" at high levels. These include components of the nucleocapsid which surround the viral DNA during the formation of progeny virus particles. Production of progeny virus particles begins around 12 hours pi. Initially, progeny virus migrate to the cell membrane where they acquire an envelope as they bud out from the surface of the cell. The nonoccluded virus particles can then infect other cells within the insect. Polyhedrin synthesis begins approximately 18 hours after infection and increases to very high levels by 24 to 48 hours pi. At about 24 hrs pi, there is a decrease in the rate of nonoccluded virus production, and most progeny virus particles are then embedded in occlusion bodies. Occlusion body formation continues until the cell dies or lyses. Some baculoviruses infect virtually every tissue in the host insect so that at the end of the infection process, the entire insect is liquified, releasing extremely large numbers of occlusion bodies which can then spread the infection to other insects. [Reviewed in The Biology of Baculoviruses, Vol. I and II, Granados and Federici (eds.), CRC Press, Boca Raton, Fla., 1986].
Baculoviruses which are derivatives of AcMNPV and are useful as expression vectors have been described in U.S. Pat. No. 5,244,805 (Miller, issued Sep. 14, 1993); Rankin et al. (1988) Gene 70:39-49; Ooi et al. (1989) J. Mol. Biol. 210:721-736, Thiem and Miller (1990) Gene 91:87-95. Particularly strong late and very late promoters include the modified polyhedrin promoter LSXIV, the hybrid Cap/Polh promoter and the synthetic promoter Syn. However, there is a need for baculoviruses which cause insects to cease feeding earlier than prior art baculoviruses so that crop damage is minimized.
Baculoviruses with improved insecticidal properties have been described. For example, AcMNPV in which the egt (ecdysone glucosyl transferase) gene has been inactivated causes earlier cessation of feeding and earlier larvae death as compared to larvae infected with wild-type AcMNPV [See, e.g., U.S. Pat. No. 5,352,451 (Miller et al., issued Oct. 4, 1994].
Pyemotes tritici, the straw-itch mite, is one of thirteen known species of mites in the genus Pyemotes, all of which are predatory and which possess venoms causing mild to extreme toxicity in target insects. The thirteen known species can be divided into two morphological groups which also differ in host preference, methods of dispersal and toxicity to their target prey, and in the effects of their toxins on insects and man. The scolyti and ventricosus groups are summarized in Table 1. Most members of the ventricosus group have extremely insect-toxic venoms. The mite venoms do not appear to be specific for particular insects, since the venoms are toxic to a wide variety of insect host and nonhost species. However, the P. tritici toxins do not appear to be toxic to mammals.
Insect-specific toxins in the venom of P. tritici, have been purified and characterized [Tomalski et al. (1988) Toxicon 26:127-132; Tomalski et al. (1989) Toxicon 27:1151-1167]. These toxins are produced in female mites and injected into insect prey as components of the venom, resulting in paralysis of the prey, which allows the feeding female mite to become fully gravid, thus ensuring adequate nutrients for reproduction. The toxin designated TxP-I has been purified to apparent homogeneity; it has an apparent molecular weight of 27,000, as determined by SDS-polyacrylamide gel electrophoresis. Two other components were resolved which exhibit molecular weights of 28,000 and 29,000; these two components comprise TXP-II. Based on peptide mapping and immunoblot experiments, it was concluded that the protein components of TxP-I and TxP-II are isoproteins [Tomalski et al. (1989) supra]. DNA sequences encoding P. tritici toxin proteins have been isolated and characterized, and expressed in AcMNPV. See, e.g., U.S. Pat. No. 5,266,317, which is incorporated by reference herein in its entirety.
Insect-specific neurotoxins have also been found in the venoms of other arthropods including, but not limited to, scorpions, wasps and spiders [Zlotkin (1985) in Comprehensive Insect Physiology, Biochemistry and Pharmacology, I. Kerkut and L. I. Gilbert (eds.) Pergamon Press, Oxford, U.K., pp. 499-546]. Several insect-specific toxins (and corresponding coding sequences) from scorpions and other insect predators have also been described [See, e.g., EP 505 207 (published Sep. 23, 1992, Cayley et al.); Maeda et al. (1991) Virology 184:777-780; McCutchen et al. (1991) Bio/Technology 9:848-852; Stewart et al. (1991) Nature 352:85-88]. Merryweather et al. (1990) J. Gen. Virology 71:1535-1544 reported the construction of baculovirus containing the Bacillus thuringiensis subsp. kurstaki HD-73 delta endotoxin expressed the control of the polyhedrin promoter.